Glucose isomerase catalyzes the reversible isomerization of glucose to fructose. Fructose is nowadays commonly applied as sugar substitute due to its higher sweetness compared to, e.g., sucrose and glucose. Many microorganisms are known to produce glucose isomerase, see, for example, the review articles by Wen-Pin Chen in Process Biochemistry, 15 June/July (1980) 30-41 and August/September (1980) 36-41, in which a large number of microorganisms capable of producing glucose isomerase are listed.
Several microorganisms have been applied industrially. The Wen-Pin Chen reference describes culture conditions of the microorganisms and recovery and purification methods of the produced glucose isomerase.
The production of glucose isomerase, which is an intracellular enzyme, is relatively expensive. Special formulations have been developed to enable repeated and continuous use of the enzyme. By immobilizing the enzyme, usually in water-insoluble form, it can be used both in batch and continuous processes (e.g., packed-bed reactors). One of the major drawbacks of immobilization of glucose isomerase is the substantial decrease of specific activity, due to the presence of inert material. The situation becomes even worse during application, since glucose isomerase is inactivated at elevated temperatures. An irreversible loss of activity will be the result of the heat-induced deterioration.
Despite efforts to retain enzyme stability substantial activity loss is still encountered under normal application conditions. There is, therefore, a continuous need for new enzymes such as glucose isomerase with improved properties. Improved thermostability of glucose isomerase, for example, will allow to take advantage of the fact that the equilibrium of the isomerisation is shifted towards fructose at higher temperatures. Most glucose isomerases are applied at pH 7.5. However, fructose is not stable at this pH. Therefore, there is also a need for glucose isomerases which can be applied below pH 7.5.
Enzymes with improved properties can be developed or found in several ways, for example by classical screening methods, by chemical modification of existing proteins, or by using modern genetic and protein engineering techniques.
Screening for organisms or microorganisms that display the desired enzymatic activity, can be performed for example by isolating and purifying the enzyme from a microorganism or from a culture supernatant of such microorganisms, determining its biochemical properties and checking whether these biochemical properties meet the demands for application.
If the identified enzyme cannot be obtained from its natural producing organism, recombinant-DNA techniques may be used to isolate the gene encoding the enzyme, express the gene in another organism, isolate and purify the expressed enzyme and test whether it is suitable for the intended application.
Modification of existing enzymes can be achieved inter alia by chemical modification methods. See, for example, I. Svendsen, Carlsberg Res. Commun. 44 (1976), 237-291. In general, these methods are too unspecific in that they modify all accessible residues with common side chains, or they are dependent on the presence of suitable amino acids to be modified, and often they are unable to modify amino acids difficult to reach, unless the enzyme molecule is unfolded. Enzyme modification through mutagenesis of the encoding gene does not suffer from the aspecificities mentioned above, and therefore is thought to be superior. Mutagenesis can be achieved either by random mutagenesis or by site-directed mutagenesis.
Random mutagenesis, by treating whole microorganisms with chemical mutagens or with mutagenizing radiation, may of course result in modified enzymes, but then strong selection protocols are necessary to search for mutants having the desired properties. Higher probability of isolating desired mutant enzymes by random mutagenesis can be achieved by cloning the encoding gene, mutagenizing it in vitro or in vivo and expressing the encoded enzyme by recloning of the mutated gene in a suitable host cell. Also in this case suitable biological selection protocols must be available in order to select the desired mutant enzymes. These biological selection protocols do not specifically select enzymes suited for application in the fructose production.
Site-directed mutagenesis (SDM) is the most specific way of obtaining modified enzymes, enabling specific substitution of one or more amino acids by any other desired amino acid.